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1.
Methods Mol Biol ; 2409: 77-96, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34709637

RESUMO

Dengue replicons are powerful tools used in studying virus biology as well as in high-throughput screening of drug candidates. Replicon constructs are developed as genomic (consists of all the viral protein genes) or sub-genomic (consists of only nonstructural protein genes) and are used to study various aspects of the virus life cycle such as genome replication and virus assembly. In addition, a replicon usually includes a reporter gene used in monitoring virus replication. In this chapter, we provide methods to develop both genomic and sub-genomic dengue replicons with a luciferase reporter and describe different assays to utilize these systems.


Assuntos
Vírus da Dengue , Dengue/genética , Vírus da Dengue/genética , Genes Reporter , Genômica , Humanos , RNA Viral , Replicon/genética , Replicação Viral/genética
2.
Int J Mol Sci ; 22(21)2021 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-34769072

RESUMO

Inhaled nebulized interferon (IFN)-α and IFN-ß have been shown to be effective in the management of coronavirus disease 2019 (COVID-19). We aimed to construct a virus-free rapid detection system for high-throughput screening of IFN-like compounds that induce viral RNA degradation and suppress the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We prepared a SARS-CoV-2 subreplicon RNA expression vector which contained the SARS-CoV-2 5'-UTR, the partial sequence of ORF1a, luciferase, nucleocapsid, ORF10, and 3'-UTR under the control of the cytomegalovirus promoter. The expression vector was transfected into Calu-3 cells and treated with IFN-α and the IFNAR2 agonist CDM-3008 (RO8191) for 3 days. SARS-CoV-2 subreplicon RNA degradation was subsequently evaluated based on luciferase levels. IFN-α and CDM-3008 suppressed SARS-CoV-2 subreplicon RNA in a dose-dependent manner, with IC50 values of 193 IU/mL and 2.54 µM, respectively. HeLa cells stably expressing SARS-CoV-2 subreplicon RNA were prepared and treated with the IFN-α and pan-JAK inhibitor Pyridone 6 or siRNA-targeting ISG20. IFN-α activity was canceled with Pyridone 6. The knockdown of ISG20 partially canceled IFN-α activity. Collectively, we constructed a virus-free rapid detection system to measure SARS-CoV-2 RNA suppression. Our data suggest that the SARS-CoV-2 subreplicon RNA was degraded by IFN-α-induced ISG20 exonuclease activity.


Assuntos
Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Interferon-alfa/farmacologia , RNA Viral/metabolismo , SARS-CoV-2/genética , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Exorribonucleases/genética , Vetores Genéticos , Células HeLa , Humanos , Interferon-alfa/administração & dosagem , Luciferases/genética , Luciferases/metabolismo , Naftiridinas/administração & dosagem , Naftiridinas/farmacologia , Oxidiazóis/administração & dosagem , Oxidiazóis/farmacologia , RNA Viral/efeitos dos fármacos , Replicon
3.
Int J Mol Sci ; 22(19)2021 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-34638661

RESUMO

Bacteria of the genus Listeria (phylum Firmicutes) include both human and animal pathogens, as well as saprophytic strains. A common component of Listeria spp. genomes are plasmids, i.e., extrachromosomal replicons that contribute to gene flux in bacteria. This study provides an in-depth insight into the structure, diversity and evolution of plasmids occurring in Listeria strains inhabiting various environments under different anthropogenic pressures. Apart from the components of the conserved plasmid backbone (providing replication, stable maintenance and conjugational transfer functions), these replicons contain numerous adaptive genes possibly involved in: (i) resistance to antibiotics, heavy metals, metalloids and sanitizers, and (ii) responses to heat, oxidative, acid and high salinity stressors. Their genomes are also enriched by numerous transposable elements, which have influenced the plasmid architecture. The plasmidome of Listeria is dominated by a group of related replicons encoding the RepA replication initiation protein. Detailed comparative analyses provide valuable data on the level of conservation of these replicons and their role in shaping the structure of the Listeria pangenome, as well as their relationship to plasmids of other genera of Firmicutes, which demonstrates the range and direction of flow of genetic information in this important group of bacteria.


Assuntos
Genes Bacterianos/genética , Listeria/genética , Plasmídeos/genética , Elementos de DNA Transponíveis/genética , Firmicutes/genética , Genoma Bacteriano/genética , Replicon/genética
4.
Proc Natl Acad Sci U S A ; 118(43)2021 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-34686605

RESUMO

Self-amplifying RNA replicons are promising platforms for vaccine generation. Their defects in one or more essential functions for viral replication, particle assembly, or dissemination make them highly safe as vaccines. We previously showed that the deletion of the envelope (E) gene from the Middle East respiratory syndrome coronavirus (MERS-CoV) produces a replication-competent propagation-defective RNA replicon (MERS-CoV-ΔE). Evaluation of this replicon in mice expressing human dipeptidyl peptidase 4, the virus receptor, showed that the single deletion of the E gene generated an attenuated mutant. The combined deletion of the E gene with accessory open reading frames (ORFs) 3, 4a, 4b, and 5 resulted in a highly attenuated propagation-defective RNA replicon (MERS-CoV-Δ[3,4a,4b,5,E]). This RNA replicon induced sterilizing immunity in mice after challenge with a lethal dose of a virulent MERS-CoV, as no histopathological damage or infectious virus was detected in the lungs of challenged mice. The four mutants lacking the E gene were genetically stable, did not recombine with the E gene provided in trans during their passage in cell culture, and showed a propagation-defective phenotype in vivo. In addition, immunization with MERS-CoV-Δ[3,4a,4b,5,E] induced significant levels of neutralizing antibodies, indicating that MERS-CoV RNA replicons are highly safe and promising vaccine candidates.


Assuntos
Infecções por Coronavirus/prevenção & controle , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , RNA Viral/administração & dosagem , Replicon , Vacinas Virais/administração & dosagem , Animais , Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/biossíntese , Infecções por Coronavirus/genética , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Vírus Defeituosos/genética , Vírus Defeituosos/imunologia , Feminino , Deleção de Genes , Genes env , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Coronavírus da Síndrome Respiratória do Oriente Médio/patogenicidade , RNA Viral/genética , RNA Viral/imunologia , Vacinas de DNA , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas Virais/genética , Vacinas Virais/imunologia , Virulência/genética , Virulência/imunologia
5.
Signal Transduct Target Ther ; 6(1): 369, 2021 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-34697295

RESUMO

The lung is the prophylaxis target against SARS-CoV-2 infection, and neutralizing antibodies are a leading class of biological products against various infectious viral pathogen. In this study, we develop a safe and cost-effective platform to express neutralizing antibody in the lung with replicating mRNA basing on alphavirus replicon particle (VRP) delivery system, to prevent SARS-CoV-2 infections. First, a modified VEEV replicon with two subgenomic (sg) promoters was engineered to translate the light and heavy chains of antibody simultaneously, for expression and assembly of neutralizing anti-SARS-CoV-2 antibody CB6. Second, the feasibility and protective efficacy of replicating mRNA against SARS-CoV-2 infection were demonstrated through both in vitro and in vivo assays. The lung target delivery with the help of VRP system resulted in efficiently block SARS-CoV-2 infection with reducing viral titer and less tissue damage in the lung of mice. Overall, our data suggests that expressing neutralizing antibodies in the lungs with the help of self-replicating mRNA could potentially be a promising prophylaxis approach against SARS-CoV-2 infection.


Assuntos
Alphavirus , Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19/terapia , Replicon , SARS-CoV-2/metabolismo , Animais , Anticorpos Neutralizantes/biossíntese , Anticorpos Neutralizantes/genética , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/genética , COVID-19/genética , COVID-19/metabolismo , Chlorocebus aethiops , Cricetinae , Feminino , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , SARS-CoV-2/genética , Células Vero
6.
G3 (Bethesda) ; 11(9)2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34544144

RESUMO

Bacterial genomes typically consist of one large chromosome, but can also include secondary replicons. These so-called multipartite genomes are scattered on the bacterial tree of life with the majority of cases belonging to Proteobacteria. Within the class gamma-proteobacteria, multipartite genomes are restricted to the two families Vibrionaceae and Pseudoalteromonadaceae. Whereas the genome of vibrios is well studied, information on the Pseudoalteromonadaceae genome is much scarcer. We have studied Pseudoalteromonadaceae with respect to the origin of the chromid, how pangene categories are distributed, how genes are expressed relative to their genomic location, and identified chromid hallmark genes. We calculated the Pseudoalteromonadaceae pangenome based on 25 complete genomes and found that core/softcore are significantly overrepresented in late replicating sectors of the chromid, regardless of how the chromid is replicated. On the chromosome, core/softcore and shell/cloud genes are only weakly overrepresented at the chromosomal replication origin and termination sequences, respectively. Gene expression is trending downwards with increasing distance from the chromosomal oriC, whereas the chromidal expression pattern is more complex. Moreover, we identified 78 chromid hallmark genes, and BLASTp searches suggest that the majority of them were acquired from the ancestral gene pool of Alteromonadales. Finally, our data strongly suggest that the chromid originates from a plasmid that was acquired in a relatively recent event. In summary, this study extends our knowledge on multipartite genomes, and helps us understand how and why secondary replicons are acquired, why they are maintained, and how they are shaped by evolution.


Assuntos
Pseudoalteromonas , Genoma Bacteriano , Genômica , Humanos , Plasmídeos , Replicon
7.
J Virol ; 95(20): e0190620, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34346768

RESUMO

Characterized positive-strand RNA viruses replicate in association with intracellular membranes. Regarding viruses in the genus Potexvirus, the mechanism by which their RNA-dependent RNA polymerase (replicase) associates with membranes is understudied. Here, by membrane flotation analyses of the replicase of Plantago asiatica mosaic potexvirus (PlAMV), we identified a region in the methyltransferase (MET) domain as a membrane association determinant. An amphipathic α-helix was predicted downstream from the core region of the MET domain, and hydrophobic amino acid residues were conserved in the helical sequences in replicases of other potexviruses. Nuclear magnetic resonance (NMR) analysis confirmed the amphipathic α-helical configuration and unveiled a kink caused by a highly conserved proline residue in the α-helix. Substitution of this proline residue and other hydrophobic and charged residues in the amphipathic α-helix abolished PlAMV replication. Ectopic expression of a green fluorescent protein (GFP) fusion with the entire MET domain resulted in the formation of a large perinuclear complex, where virus replicase and RNA colocated during virus infection. Except for the proline substitution, the amino acid substitutions in the α-helix that abolished virus replication also prevented the formation of the large perinuclear complex by the respective GFP-MET fusion. Small intracellular punctate structures were observed for all GFP-MET fusions, and in vitro high-molecular-weight complexes were formed by both replication-competent and -incompetent viral replicons and thus were not sufficient for replication competence. We discuss the roles of the potexvirus-specific, proline-kinked amphipathic helical structure in virus replication and intracellular large complex and punctate structure formation. IMPORTANCE RNA viruses characteristically associate with intracellular membranes during replication. Although virus replicases are assumed to possess membrane-targeting properties, their membrane association domains generally remain unidentified or poorly characterized. Here, we identified a proline-kinked amphipathic α-helix structure downstream from the methyltransferase core domain of PlAMV replicase as a membrane association determinant. This helical sequence, which includes the proline residue, was conserved among potexviruses and related viruses in the order Tymovirales. Substitution of the proline residue, but not the other residues necessary for replication, allowed formation of a large perinuclear complex within cells resembling those formed by PlAMV replicase and RNA during virus replication. Our results demonstrate the role of the amphipathic α-helix in PlAMV replicase in a perinuclear complex formation and virus replication and that perinuclear complex formation by the replicase alone will not necessarily indicate successful virus replication.


Assuntos
Potexvirus/genética , Potexvirus/metabolismo , Proteínas do Complexo da Replicase Viral/genética , Sequência de Aminoácidos/genética , Proteínas de Membrana/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismo , Doenças das Plantas/virologia , Prolina/genética , RNA Viral/genética , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Replicon/genética , Tabaco/virologia , Proteínas Virais/metabolismo , Proteínas do Complexo da Replicase Viral/metabolismo , Replicação Viral/genética
8.
Microb Pathog ; 159: 105124, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34364978

RESUMO

OBJECTIVES: Pseudomonas aeruginosa is a medically important pathogen showing intrinsic low permeability to various antimicrobial agents and its potential to acquire multiple resistance mechanism. A longitudinal surveillance aimed to investigate the antimicrobial resistance and its determinants of Pseudomonas aeruginosa in Southern China. A total of 2163 P. aeruginosa isolates were obtained from patients in Southern China during 2004-2016. METHODS: The antimicrobial susceptibility of the isolates was performed by disk diffusion and Vitek 2 automated system and interpreted according to the Clinical and Laboratory Standard Institute (CLSI) 2015. RESULTS: A significant downtrend of resistant rate (>10.0%) was observed for tested antibiotic agents including ciprofloxacin (>30.0%), gentamicin (29.0%), tobramycin (24.2%) and ceftazidime (24.0%) except for aztreonam and amikacin. A total of 269 randomly selected isolates were further studied on the carriage of ß-lactam resistance genes by using 7 groups of multiplex PCRs targeting on 20 genes. ß-lactam resistance genes were rarely detected with a rate lower than 8%. Among all ß-lactam resistance genes, blaSHV acquired the highest identification rate (18/269, 6.7%), followed by blaOXA-1-like (6/269, 2.2%) and blaPER (6/269, 2.2%). In addition, 8 different plasmid replicons were amplified using 8 groups of multiplex PCRs including 18 sets of primers. Only five plasmid replicons were identified in 5 different P. aeruginosa isolates. Insignificant clonal relatedness among the positive strains identified by regular PCR were further verified by randomly amplified polymorphic DNA (RAPD)-PCR. CONCLUSION: This study has provided comprehensive knowledge on current antimicrobial resistance, ß-lactam resistance genes and plasmid replicons carriage in a large scale of clinical P. aeruginosa isolates.


Assuntos
Infecções por Pseudomonas , Pseudomonas aeruginosa , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Farmacorresistência Bacteriana , Humanos , Testes de Sensibilidade Microbiana , Plasmídeos/genética , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/genética , Técnica de Amplificação ao Acaso de DNA Polimórfico , Replicon , beta-Lactamases/genética
9.
J Virol ; 95(19): e0068621, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34232709

RESUMO

During persistent human papillomavirus infection, the viral genome replicates as an extrachromosomal plasmid that is efficiently partitioned to daughter cells during cell division. We have previously shown that an element which overlaps the human papillomavirus 18 (HPV18) transcriptional enhancer promotes stable DNA replication of replicons containing the viral replication origin. Here, we perform comprehensive analyses to elucidate the function of this maintenance element. We conclude that no unique element or binding site in this region is absolutely required for persistent replication and partitioning and instead propose that the overall chromatin architecture of this region is important to promote efficient use of the replication origin. These results have important implications for the genome partitioning mechanism of papillomaviruses. IMPORTANCE Persistent infection with oncogenic human papillomaviruses (HPVs) is responsible for ∼5% of human cancers. The viral DNA replicates as an extrachromosomal plasmid and is partitioned to daughter cells in dividing keratinocytes. Using a complementation assay that allows us to separate viral transcription and replication, we provide insight into viral sequences that are required for long-term replication and persistence in keratinocytes. Understanding how viral genomes replicate persistently for such long periods of time will guide the development of antiviral therapies.


Assuntos
Genoma Viral , Papillomavirus Humano 18/genética , Papillomavirus Humano 18/fisiologia , Sequências Reguladoras de Ácido Nucleico , Replicon/fisiologia , Replicação Viral , Sítios de Ligação , Cromatina/fisiologia , Replicação do DNA , Elementos Facilitadores Genéticos , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/fisiologia , Papillomavirus Humano 31/genética , Papillomavirus Humano 31/fisiologia , Queratinócitos/fisiologia , Queratinócitos/virologia , Plasmídeos , Regiões Promotoras Genéticas , Origem de Replicação , Fator de Transcrição AP-1/metabolismo , Transcrição Genética
10.
J Virol ; 95(18): e0068721, 2021 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-34191580

RESUMO

The emerging coronavirus disease 2019 (COVID-19) outbreak caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread worldwide, resulting in global public health emergencies and economic crises. In the present study, a noninfectious and biosafety level 2 (BSL2)-compatible SARS-CoV-2 replicon expressing a nano luciferase (nLuc) reporter was constructed in a bacterial artificial chromosomal (BAC) vector by reverse genetics. The nLuc reporter is highly sensitive, easily quantifiable, and high throughput adaptable. Upon transfecting the SARS-CoV-2 replicon BAC plasmid DNA into Vero E6 cells, we could detect high levels of nLuc reporter activity and viral RNA transcript, suggesting the replication of the replicon. The replicon replication was further demonstrated by the findings that deleting nonstructural protein 15 or mutating its catalytic sites significantly reduced replicon replication, whereas providing the nucleocapsid protein in trans enhanced replicon replication in a dose-dependent manner. Finally, we showed that remdesivir, a U.S. Food and Drug Administration-approved antiviral drug, significantly inhibited the replication of the replicon, providing proof of principle for the application of our replicon as a useful tool for developing antivirals. Taken together, this study established a sensitive and BSL2-compatible reporter system in a single BAC plasmid for investigating the functions of SARS-CoV-2 proteins in viral replication and evaluating antiviral compounds. This should contribute to the global effort to combat this deadly viral pathogen. IMPORTANCE The COVID-19 pandemic caused by SARS-CoV-2 is having a catastrophic impact on human lives. Combatting the pandemic requires effective vaccines and antiviral drugs. In the present study, we developed a SARS-CoV-2 replicon system with a sensitive and easily quantifiable reporter. Unlike studies involving infectious SARS-CoV-2 virus that must be performed in a biosafety level 3 (BSL3) facility, the replicon is noninfectious and thus can be safely used in BSL2 laboratories. The replicon will provide a valuable tool for testing antiviral drugs and studying SARS-CoV-2 biology.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Avaliação Pré-Clínica de Medicamentos , Proteínas de Fluorescência Verde/metabolismo , Replicon , SARS-CoV-2/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , COVID-19/virologia , Chlorocebus aethiops , Proteínas de Fluorescência Verde/genética , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos
11.
J Gen Virol ; 102(5)2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33956592

RESUMO

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus, which is highly pathogenic and classified as a biosafety level 3 (BSL-3) agent, has greatly threatened global health and efficacious antivirals are urgently needed. The high requirement of facilities to manipulate the live virus has limited the development of antiviral study. Here, we constructed a reporter replicon of SARS-CoV-2, which can be handled in a BSL-2 laboratory. The Renilla luciferase activity effectively reflected the transcription and replication levels of the replicon genome. We identified the suitability of the replicon in antiviral screening using the known inhibitors, and thus established the replicon-based high-throughput screening (HTS) assay for SARS-CoV-2. The application of the HTS assay was further validated using a few hit natural compounds, which were screened out in a SARS-CoV-2 induced cytopathic-effect-based HTS assay in our previous study. This replicon-based HTS assay will be a safe platform for SARS-CoV-2 antiviral screening in a BSL-2 laboratory without the live virus.


Assuntos
Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Replicon/efeitos dos fármacos , SARS-CoV-2/efeitos dos fármacos , Animais , COVID-19/tratamento farmacológico , Chlorocebus aethiops , Descoberta de Drogas , Ensaios de Triagem em Larga Escala/métodos , Humanos , Replicon/genética , SARS-CoV-2/genética , Células Vero , Replicação Viral/efeitos dos fármacos
12.
J Virol ; 95(14): e0058921, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-33952642

RESUMO

Negative-stranded RNA (NSR) viruses include both animal- and plant-infecting viruses that often cause serious diseases in humans and livestock and in agronomic crops. Rice stripe tenuivirus (RSV), a plant NSR virus with four negative-stranded/ambisense RNA segments, is one of the most destructive rice pathogens in many Asian countries. Due to the lack of a reliable reverse-genetics technology, molecular studies of RSV gene functions and its interaction with host plants are severely hampered. To overcome this obstacle, we developed a mini-replicon-based reverse-genetics system for RSV gene functional analysis in Nicotiana benthamiana. We first developed a mini-replicon system expressing an RSV genomic RNA3 enhanced green fluorescent protein (eGFP) reporter [MR3(-)eGFP], a nucleocapsid (NP), and a codon usage-optimized RNA-dependent RNA polymerase (RdRpopt). Using this mini-replicon system, we determined that RSV NP and RdRpopt are indispensable for the eGFP expression from MR3(-)eGFP. The expression of eGFP from MR3(-)eGFP can be significantly enhanced in the presence of four viral suppressors of RNA silencing (VSRs), NSs, and P19-HcPro-γb. In addition, NSvc4, the movement protein of RSV, facilitated eGFP trafficking between cells. We also developed an antigenomic RNA3-based replicon in N. benthamiana. However, we found that the RSV NS3 coding sequence acts as a cis element to regulate viral RNA expression. Finally, we made mini-replicons representing all four RSV genomic RNAs. This is the first mini-replicon-based reverse-genetics system for monocot-infecting tenuivirus. We believe that the mini-replicon system described here will allow studies of the RSV replication, transcription, cell-to-cell movement, and host machinery underpinning RSV infection in plants. IMPORTANCE Plant-infecting segmented negative-stranded RNA (NSR) viruses are grouped into three genera: Orthotospovirus, Tenuivirus, and Emaravirus. Reverse-genetics systems have been established for members of the genera Orthotospovirus and Emaravirus. However, there is still no reverse-genetics system available for Tenuivirus. Rice stripe virus (RSV) is a monocot-infecting tenuivirus with four negative-stranded/ambisense RNA segments. It is one of the most destructive rice pathogens and causes significant damage to the rice industry in Asian countries. Due to the lack of a reliable reverse-genetics system, molecular characterizations of RSV gene functions and the host machinery underpinning RSV infection in plants are extremely difficult. To overcome this obstacle, we developed a mini-replicon-based reverse-genetics system for RSV in Nicotiana benthamiana. This is the first mini-replicon-based reverse-genetics system for tenuivirus. We consider that this system will provide researchers a new working platform to elucidate the molecular mechanisms dictating segmented tenuivirus infections in plants.


Assuntos
Genes Fúngicos/fisiologia , Replicon , Genética Reversa , Tenuivirus/genética , Tabaco/virologia , Regulação Viral da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Movimento , Nucleocapsídeo/genética , Interferência de RNA , Proteínas não Estruturais Virais/genética
13.
Virol Sin ; 36(5): 913-923, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33999369

RESUMO

SARS-CoV-2 causes the pandemic of COVID-19 and no effective drugs for this disease are available thus far. Due to the high infectivity and pathogenicity of this virus, all studies on the live virus are strictly confined in the biosafety level 3 (BSL3) laboratory but this would hinder the basic research and antiviral drug development of SARS-CoV-2 because the BSL3 facility is not commonly available and the work in the containment is costly and laborious. In this study, we constructed a reverse genetics system of SARS-CoV-2 by assembling the viral cDNA in a bacterial artificial chromosome (BAC) vector with deletion of the spike (S) gene. Transfection of the cDNA into cells results in the production of an RNA replicon that keeps the capability of genome or subgenome replication but is deficient in virion assembly and infection due to the absence of S protein. Therefore, such a replicon system is not infectious and can be used in ordinary biological laboratories. We confirmed the efficient replication of the replicon by demonstrating the expression of the subgenomic RNAs which have similar profiles to the wild-type virus. By mutational analysis of nsp12 and nsp14, we showed that the RNA polymerase, exonuclease, and cap N7 methyltransferase play essential roles in genome replication and sgRNA production. We also created a SARS-CoV-2 replicon carrying a luciferase reporter gene and this system was validated by the inhibition assays with known anti-SARS-CoV-2 inhibitors. Thus, such a one-plasmid system is biosafe and convenient to use, which will benefit both fundamental research and development of antiviral drugs.


Assuntos
Antivirais , COVID-19 , Antivirais/farmacologia , Contenção de Riscos Biológicos , Humanos , Replicon , SARS-CoV-2 , Replicação Viral
14.
J Med Microbiol ; 70(5)2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33999798

RESUMO

Introduction. New Delhi metallo-ß-lactamase (NDM)-producing Klebsiella pneumoniae has become a serious global health concern.Hypothesis/Gap Statement. Due to the high genetic diversity among NDM-positive K. pneumoniae, we need further surveillance and studies to better understand the relationships between them. In addition, the coexistence of several plasmid replicon types in NDM-positive K. pneumoniae may affect the copy number of bla NDM, the MIC level to antibiotics, as well as increasing the chance of horizontal gene transfer.Aim. The aim of this study was to determine incompatible plasmid groups and copy numbers of bla NDM, and to investigate the genetic relationship of 37 NDM-positive K. pneumoniae in Kerman, Iran.Methodology. The bla NDM-1 gene was detected and confirmed by PCR-sequencing. The plasmid replicon types were determined by PCR-based replicon typing (PBRT) and the copy number of bla NDM-1 was determined by quantitaive real time-PCR (qPCR). Random amplified polymorphic DNA (RAPD)-PCR typing was used to detect genetic relationships between the strains.Results. In this study, 10 different replicon types, including Frep [n=25 (67.5 %)], FIIAs [n=11 (29.7 %)], FIA [n=5 (13.5 %)], FIB [n=3 (8.1 %)], I1-Iγ [n=2 (5.4 %)], L/M [n=7 (18.9 %)], A/C [n=7 (18.9 %)], Y [n=3 (8.1 %)], P [n=1 (2.7 %)] and FIC [n=1 (2.7 %)] were reported. The copy numbers of the bla NDM-1 gene varied from 30.00 to 5.0×106 and no statistically significant correlation was observed between a rise of the MIC to imipenem and the copy numbers of bla NDM-1 (P>0.05). According to RAPD typing results, 35 strains were divided into five clusters, while two strains were non-typeable.Conclusion. The spread of NDM-1-producing K. pneumoniae strains that carry several plasmid replicon types increases the chance of horizontal transfer of antibiotic resistance genes in hospital settings. In this study, 10 different replicon types were identified. We could not find any relationship between the increase of MIC levels to imipenem and the copy numbers of bla NDM-1. Therefore, due to the identification of different replicon types in this study, the type and genetic characteristics of bla NDM-1-carrying plasmids, and other factors such as antibiotic selective pressure, probably affect the copy number of bla NDM-1 and change the MIC level to imipenem.


Assuntos
Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Dosagem de Genes , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/genética , Plasmídeos , beta-Lactamases/genética , Humanos , Irã (Geográfico) , Klebsiella pneumoniae/classificação , Tipagem Molecular , Técnica de Amplificação ao Acaso de DNA Polimórfico , Replicon , Resistência beta-Lactâmica
15.
Mol Ther ; 29(6): 1970-1983, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-33823303

RESUMO

A self-transcribing and replicating RNA (STARR)-based vaccine (LUNAR-COV19) has been developed to prevent SARS-CoV-2 infection. The vaccine encodes an alphavirus-based replicon and the SARS-CoV-2 full-length spike glycoprotein. Translation of the replicon produces a replicase complex that amplifies and prolongs SARS-CoV-2 spike glycoprotein expression. A single prime vaccination in mice led to robust antibody responses, with neutralizing antibody titers increasing up to day 60. Activation of cell-mediated immunity produced a strong viral antigen-specific CD8+ T lymphocyte response. Assaying for intracellular cytokine staining for interferon (IFN)γ and interleukin-4 (IL-4)-positive CD4+ T helper (Th) lymphocytes as well as anti-spike glycoprotein immunoglobulin G (IgG)2a/IgG1 ratios supported a strong Th1-dominant immune response. Finally, single LUNAR-COV19 vaccination at both 2 µg and 10 µg doses completely protected human ACE2 transgenic mice from both mortality and even measurable infection following wild-type SARS-CoV-2 challenge. Our findings collectively suggest the potential of LUNAR-COV19 as a single-dose vaccine.


Assuntos
Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/biossíntese , Vacinas contra COVID-19/administração & dosagem , COVID-19/prevenção & controle , SARS-CoV-2/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Sintéticas/administração & dosagem , Alphavirus/genética , Alphavirus/imunologia , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/imunologia , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , COVID-19/imunologia , COVID-19/patologia , COVID-19/virologia , Vacinas contra COVID-19/biossíntese , Vacinas contra COVID-19/genética , Vacinas contra COVID-19/imunologia , Feminino , Expressão Gênica , Humanos , Imunidade Celular/efeitos dos fármacos , Imunidade Humoral/efeitos dos fármacos , Interferon gama/genética , Interferon gama/imunologia , Interleucina-4/genética , Interleucina-4/imunologia , Camundongos , Camundongos Transgênicos , Replicon/imunologia , SARS-CoV-2/imunologia , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th1/virologia , Transgenes , Resultado do Tratamento , Vacinação/métodos , Vacinas Sintéticas/biossíntese , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia
16.
J Exp Bot ; 72(13): 4796-4808, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-33872346

RESUMO

Rapeseed (Brassica napus L.) is an important oil crop worldwide, and effective weed control can protect its yield and quality. Farmers can benefit from cultivars tolerant to herbicides such as glyphosate. Amino acid substitutions in enolpyruvylshikimate-3-phosphate synthase (EPSPS) render the plant less sensitive to glyphosate. Therefore, we aimed to optimize the glyphosate tolerance trait in rapeseed via endogenous EPSPS modification. To achieve effective gene replacement in B. napus L., we employed a CRISPR/Cas9 system expressing single-guide RNAs (sgRNAs) cleaved by the CRISPR-associated RNA endoribonuclease Csy4 from Pseudomonas aeruginosa, for targeted induction of double-strand breaks. Both the donor template and a geminiviral replicon harbouring an sgRNA expression cassette were introduced into plant cells. Using sgRNAs targeting adjacent donor DNA template containing synonymous mutations in sgRNA sites, we achieved precise gene replacements in the endogenous B. napus EPSPS gene, BnaC04EPSPS, resulting in amino acid substitutions at frequencies up to 20%. Rapeseed seedlings harbouring these substitutions were glyphosate-tolerant. Furthermore, modifications in BnaC04EPSPS were precisely transmitted to the next generation. Our genome editing strategy enables highly efficient gene targeting and the induction of glyphosate tolerance in oilseed rape.


Assuntos
Brassica napus , RNA Guia , Brassica napus/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA , Glicina/análogos & derivados , Processamento Pós-Transcricional do RNA , Replicon
17.
Virol Sin ; 36(5): 890-900, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33835389

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a devastating pandemic worldwide. Vaccines and antiviral drugs are the most promising candidates for combating this global epidemic, and scientists all over the world have made great efforts to this end. However, manipulation of the SARS-CoV-2 should be performed in the biosafety level 3 laboratory. This makes experiments complicated and time-consuming. Therefore, a safer system for working with this virus is urgently needed. Here, we report the construction of plasmid-based, non-infectious SARS-CoV-2 replicons with turbo-green fluorescent protein and/or firefly luciferase reporters by reverse genetics using transformation-associated recombination cloning in Saccharomyces cerevisiae. Replication of these replicons was achieved simply by direct transfection of cells with the replicon plasmids as evident by the expression of reporter genes. Using SARS-CoV-2 replicons, the inhibitory effects of E64-D and remdesivir on SARS-CoV-2 replication were confirmed, and the half-maximal effective concentration (EC50) value of remdesivir and E64-D was estimated by different quantification methods respectively, indicating that these SARS-CoV-2 replicons are useful tools for antiviral drug evaluation.


Assuntos
COVID-19 , SARS-CoV-2 , Antivirais/farmacologia , Avaliação de Medicamentos , Humanos , Replicon , Replicação Viral
18.
Proc Natl Acad Sci U S A ; 118(15)2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33766889

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research and antiviral discovery are hampered by the lack of a cell-based virus replication system that can be readily adopted without biosafety level 3 (BSL-3) restrictions. Here, the construction of a noninfectious SARS-CoV-2 reporter replicon and its application in deciphering viral replication mechanisms and evaluating SARS-CoV-2 inhibitors are presented. The replicon genome is replication competent but does not produce progeny virions. Its replication can be inhibited by RdRp mutations or by known SARS-CoV-2 antiviral compounds. Using this system, a high-throughput antiviral assay has also been developed. Significant differences in potencies of several SARS-CoV-2 inhibitors in different cell lines were observed, which highlight the challenges of discovering antivirals capable of inhibiting viral replication in vivo and the importance of testing compounds in multiple cell culture models. The generation of a SARS-CoV-2 replicon provides a powerful platform to expand the global research effort to combat COVID-19.


Assuntos
Antivirais/farmacologia , COVID-19/virologia , Ensaios de Triagem em Larga Escala/métodos , Replicon/efeitos dos fármacos , SARS-CoV-2/efeitos dos fármacos , Células A549 , Animais , Chlorocebus aethiops , RNA-Polimerase RNA-Dependente de Coronavírus/genética , Células HEK293 , Humanos , Replicon/genética , SARS-CoV-2/genética , Células Vero , Replicação Viral/efeitos dos fármacos
19.
Int J Mol Sci ; 22(3)2021 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-33573129

RESUMO

The Lactococcus lactis bacterium found in different natural environments is traditionally associated with the fermented food industry. But recently, its applications have been spreading to the pharmaceutical industry, which has exploited its probiotic characteristics and is moving towards its use as cell factories for the production of added-value recombinant proteins and plasmid DNA (pDNA) for DNA vaccination, as a safer and industrially profitable alternative to the traditional Escherichia coli host. Additionally, due to its food-grade and generally recognized safe status, there have been an increasing number of studies about its use in live mucosal vaccination. In this review, we critically systematize the plasmid replicons available for the production of pharmaceutical-grade pDNA and recombinant proteins by L. lactis. A plasmid vector is an easily customized component when the goal is to engineer bacteria in order to produce a heterologous compound in industrially significant amounts, as an alternative to genomic DNA modifications. The additional burden to the cell depends on plasmid copy number and on the expression level, targeting location and type of protein expressed. For live mucosal vaccination applications, besides the presence of the necessary regulatory sequences, it is imperative that cells produce the antigen of interest in sufficient yields. The cell wall anchored antigens had shown more promising results in live mucosal vaccination studies, when compared with intracellular or secreted antigens. On the other side, engineering L. lactis to express membrane proteins, especially if they have a eukaryotic background, increases the overall cellular burden. The different alternative replicons for live mucosal vaccination, using L. lactis as the DNA vaccine carrier or the antigen producer, are critically reviewed, as a starting platform to choose or engineer the best vector for each application.


Assuntos
Reatores Biológicos/microbiologia , Vetores Genéticos/genética , Microbiologia Industrial/métodos , Lactococcus lactis/genética , Plasmídeos/genética , Administração através da Mucosa , Engenharia Celular/métodos , DNA Circular/biossíntese , DNA Circular/genética , DNA Circular/isolamento & purificação , Tecnologia de Alimentos/métodos , Engenharia Genética/métodos , Lactococcus lactis/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Replicon/genética , Tecnologia Farmacêutica/métodos , Vacinas de DNA/administração & dosagem , Vacinas de DNA/biossíntese , Vacinas de DNA/genética , Vacinas de DNA/isolamento & purificação
20.
Virol Sin ; 36(4): 730-735, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33616893

RESUMO

Middle East respiratory syndrome coronavirus (MERS-CoV) is the causative agent of a severe respiratory disease with a high mortality of ~ 35%. The lack of approved treatments for MERS-CoV infection underscores the need for a user-friendly system for rapid drug screening. In this study, we constructed a MERS-CoV replicon containing the Renilla luciferase (Rluc) reporter gene and a stable luciferase replicon-carrying cell line. Using this cell line, we showed that MERS-CoV replication was inhibited by combined application of lopinavir and ritonavir, indicating that this cell line can be used to screen inhibitors of MERS-CoV replication. Importantly, the MERS-replicon cell line can be used for high-throughput screening of antiviral drugs without the need for live virus handling, providing an effective and safe tool for the discovery of antiviral drugs against MERS-CoV.


Assuntos
Antivirais , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Coronavírus da Síndrome Respiratória do Oriente Médio , Antivirais/farmacologia , Infecções por Coronavirus/tratamento farmacológico , Humanos , Lopinavir/uso terapêutico , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Replicon
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